DXersaid

Health
Canadian Officials Did Research On Anthrax Before U.S. Attacks
By CHAD TERHUNE, Staff Reporter of THE WALL STREET JOURNAL

ATLANTA — Canadian defense officials knew several months before the U.S. anthrax attacks that unopened envelopes containing anthrax posed health risks to mail handlers, and that opening such a letter could instantly release millions of deadly spores into the air.

They tried to warn U.S. health officials once the attacks began. But the Centers for Disease Control and Prevention didn’t open the e-mail; officials there learned the details of the Canadian study only last month.

CDC officials said last night that while the information was “relevant,” it wouldn’t have altered the way they dealt with the attacks.

Canadian officials started researching the issue in February after an anthrax hoax letter was mailed in Canada in late January; they had the basic findings by March. Researchers put a more benign but similar bacterium, Bacillus globigii, in an envelope as a surrogate for the deadly Bacillus anthracis. During a series of tests, a lab technician seated at a desk sliced open a contaminated envelope inside an aerosol test chamber and millions of spores were instantly detected. Health authorities have generally believed 8,000 to 10,000 spores are enough to infect a person with inhalational anthrax; they now think a smaller number could be enough.

“If the envelope was not completely sealed, it could also pose a threat to individuals in the mail handling system,” said the Canadian research report, dated September 2001.

The Canadian study was discussed publicly for the first time Tuesday at a CDC meeting called to discuss the anthrax attacks. Canadian officials said they e-mailed the study to the CDC Oct. 4 after anthrax first surfaced at the headquarters of a Florida tabloid. But the e-mail to an official with CDC’s laboratory response network was never opened, said Bradley Perkins, a lead anthrax investigator for the CDC. Dr. Perkins said he eventually learned of the research from a university health official around Oct. 30 and invited the Canadian researchers to make a full presentation of their study in Atlanta Nov. 4.

When the letter to Sen. Tom Daschle (D., S.D.) was discovered in October, Canadian researchers suspected postal workers might be at risk, said Bill Kournikakis, head of the preventive medicine group for chemical and biological defense at the Defence Research Establishment in Alberta, Canada, which conducted the study. But Mr. Kournikakis said he never followed up on his e-mail. Mr. Kournikakis said even if the CDC was aware of his research it was a “difficult call” about whether to give hundreds of postal workers antibiotics, because the Daschle letter was heavily taped on the ends — a scenario his study didn’t explore.

The CDC’s Dr. Perkins said he was sorry to hear the e-mail was never opened. “It is certainly relevant data, but I don’t think it would have altered the decisions that we made,” he said. He said the CDC didn’t think there was a risk to postal workers in the Washington area because no anthrax disease was found among Florida postal workers in the initial cases. “I think more weight would have been put on the Florida experience and the absence of demonstrated risk [among postal workers] than the experimental data,” he said.

Two Washington postal workers died of inhalation anthrax on Oct. 21 and 22.

Gerry Kreienkamp, a spokesman for the U.S. Postal Service, said the agency has relied on the CDC’s advice, and whether it would have responded differently depended on whether the CDC would have given “different advice to us if they had other information. We’re not in the public-health field and we don’t keep up with the latest research,” he said.

The Canadian study showed that “a lethal dose could be inhaled within seconds of opening an anthrax spore filled envelope,” said Mr. Kournikakis. “This is an aerosol that will travel through the room and get into the ventilation system.”

He said his agency’s tests showed that anthrax spores could spill from a sealed envelope and pose a health risk to postal workers. The Canadian test envelopes were sealed normally and had small openings on the edges where there is typically no adhesive. The Daschle letter was heavily sealed with tape, prompting officials at the time to think contamination from the letter was highly unlikely.

DXersaid

Many years ago I explained my view that understanding the Canadian study is important to understand why the anthrax was sent.

In mid-January 2001, it was announced that the former boss of al-Hawsawi, the guy with the anthrax spraydrying documents on his laptop, was going to have a bail hearing. His name was Mahjoub. Ayman Zawahiri was #1. Mahjoub was #2. The group was the Egyptian Islamic Jihad but with a cooler name — the Vanguards of Conquest. A letter was received January 30, 2001 at the Citizenship and Immigration Office threatening to use anthrax. It was sent to Immigration Minister Elinor Caplan who had cosigned the detention certificate. Authorities suspected that the letter was sent by militant islamists in protest over the detention of Mahjoub, who ran Bin Laden’s farm in Sudan. Mahjoub had been sentenced in absentia to 15 years in prison in 1999 by Egyptian authorities for his involvement in Egyptian Islamic Jihad. Now, he was being detained without charges under an order cosigned by Immigration Minister Caplan and threatened with deportation. The postmark has never been publicly identified. Separately, hoax letters were also sent to American businesses and a Walmart in Saanich, British Columbia. Mahjoub had been in regular contact with a man named Marzouk, who had trained the 1998 embassy bombers and was captured in Baku, Azerbaijan in August 1998.

When the letter was received in January 2001, the letter was sent by Department of National Defence jet to the Canadian Science Center for Human and Animal Health in Winnipeg for examination. Authorities also sent the filters from the Jean Edmonds building’s ventilation system. Authorities said they were treating it as a possible terrorist act against the department and noted that it “was the first time a government department has been targeted in this way.” The Ottawa alert came after one of the employees working in the Minister’s office opened a plain white envelope at 11:15 a.m. The employee discovered powder and a piece of paper in the envelope. Police refused to reveal from where it had been mailed. One source said the letter was unsigned and “mostly gibberish.” (Indeed, the Fall 2001 letters might be described as mostly gibberish, and certainly the “JLo letter” — talking about Jennifer Lopez’ planned wedding — could be.) An internal government memo distributed to staff said “an initial analysis of the envelope revealed some traces of bacteria.”

Bill Patrick, who often worked with George Mason University students in northern Virginia, had written a report in 1999 for a consultant SAIC at the request of Dr. Steve Hatfill. As one bioterrorism expert commented about the report: “Anytime you pick something up like this, and it seems to layout the whole story for you months or years before the fact, your immediate response is to step back and say ‘whoa, something may be going on here. “Our attacker may very well have used this report as something of a — if not a template, then certainly as a rule of thumb.”

After the January 2001 anthrax threat, Canadian defense research team undertook to assess the risk. The report titled “Risk Assessment of Anthrax Threat Letters” issued September 2001. In contrast to the 1998 study by William Patrick that had been requested by Dr. Hatfill’s employer SAIC, the Canadian study found considerable exposure to those in the room resulted when such a letter was opened. Bacillus globigii spores (in dry powder form) were donated by the US Department of Defense (Dugway Proving Ground, Utah). Stock concentration powder was -1 x 10 11 cfu/gm. The anthrax sent to the Senators had a smaller particle size –tending toward a uniform 1 micron, subject to clumping that easily broke apart. Bacillus globigii (BG) spores are routinely used as a simulant for Bacillus anthracis (anthrax) spores. “The letter was prepared by putting BG spores in the center of a sheet of paper, folding it over into thirds, placing the folded sheet into the envelope and sealing using the adhesive present on the envelope. The envelope was then shaken to mimic the handling and tumbling that would occur during its passage through the postal system.” The aerosol, produced by opening the BG spore containing envelope, was not confined to the area of the desk but spread throughout the chamber. Values were almost as high at the opposite end of the chamber, shortly after opening the envelopes. 99% of the particles collected were in the 2.5 to 10 mm size range. The report explained: “In addition, the aerosol would quickly spread throughout the room so that other workers, depending on their exact locations and the directional air flow within the office, would likely inhale lethal doses. Envelopes with the open corners not specifically sealed could also pose a threat to individuals in the mail handling system.”

More than 80% of the B anthracis particles collected on stationary monitors were within an alveolar respirable size range of 0.95 to 3.5 µm. Thus, the simulant performed very well. Those who continue to argue that the Daschle product was so advanced beyond what the US could do are mistaken. Indeed, the more notable question is why such a good product was prepared in response to a threat letter sent to an immigration minister. The reason perhaps is that authorities knew that it was Al Qaeda and Egyptian Islamic Jihad that sent the letter. The CIA and CSIS apparently feared that the Vanguards of Conquest would use the good stuff.

The CIA knew EIJ intended to use anthrax — from the proclamations of Jaballah’s friend, the captured military commander Mabruk and Jaballah’s brother-in-law’s former law partner al-Zayat. Authorities knew Al Qaeda was getting technical assistance from scientists — and that many of the senior Egyptian leaders had advanced or technical degrees. The specifications provided by Dugway perhaps involved treated fumed silica and a spraydryer (with a last critical step reserved to be done at Dugway) likely were based on what Al Qaeda might send with a little help from their friends.

Canadian officials explained they e-mailed the study to the CDC soon after reports of the discovery of anthrax at the American Media Inc. headquarters in Florida. The e-mail, however, was never opened, reports the lead CDC anthrax investigator, who regrets that he never read the email. “It is certainly relevant data, but I don’t think it would have altered the decisions that we made.” At one point, about 2,000 CDC employees were working on the anthrax matter. This Canadian report was perhaps the single most important scientific data point for the CDC to take into account. It certainly was one of the most important reports for the FBI to take into account. Yet I dare anyone to ask US Attorney Jeffrey Taylor if he has ever read it. Bail was denied by decision on October 5, 2001. Then highly potent anthrax was sent the next day just as had been promised. But Ayman had returned to the target of his greatest interest — rather than a Canadian immigration minister, he and Shehata and their colleagues targeted the minister who oversaw the Department of Justice and appropriations to Egypt and Israel, and who gave his name (”the Leahy Law”) to the law that permits continuing appropriations to Egypt in the face of allegations of torture. Zawahiri never makes a threat he doesn’t intend to try to keep.

The Canadian experiments in 2001 showed that if anthrax spores were finely powdered, a letter could release thousands of lethal doses of the bacteria within minutes of being opened. Furthermore, large amounts of material leaked out of sealed envelopes even before they were opened. By then, more than two dozen federal government employees knew of the Canadian studies, which showed that a real anthrax threat letter was a far more dangerous weapon than anyone had believed. Within days, a dozen more people were informed of the now highly relevant experimental findings. One FBI squad was focused on people who may have known of the study — such as William Patrick’s friend, Dr. Steve Hatfill. Another squad would be focused on the usual suspects and their friends. For the next seven years, the investigation would be shrouded in great secrecy.

The learned authors in this article discuss microencapsulation being done at Dugway.

Although it has never been mentioned, Dr. Ivins had also discussed microencapsulation done in connection with vaccines at Southern Research Institute.

Microencapsulation of pathogens in US biodefense research

Although there is no evidence to indicate that the tin and silicon content of the spores conferred any benefit for purposes of the letter attacks, their presence is meaningful if the attack spores had been prepared legitimately for other purposes. Silicone microencapsulation would have been desirable for increasing the resistance of the spores to inactivation by hazards such as UV light, ozone or toxic materials38, and for preventing detection of the spores by some methods. (It is at the spore coat, rather than the external membrane (the exosporium) of Bacillus anthracis that these functions occur39). These are properties of military concern. The use of microencapsulation for such purposes was already an old idea40 in the biodefense community in the years immediately preceding the attacks. Microencapsulation by special polymers to produce particles in the 1-10 micron range could protect microbes from environmental damage during aerosolization and delivery [e.g. via bomblets] and also from the body’s initial defenses during the infection process, according to an encyclopedia on weapons of mass destruction published in 2004, and could also help defeat some detection schemes41. The encyclopedia notes that the technology requires an advanced research and development infrastructure, unlikely to be available to terrorists, but “state-level CBW programs could certainly employ microencapsulation to produce highly effective weapons of mass destruction42”.

In the non-military literature it has recently been shown that single, living Bacillus spores can be encapsulated using layer-by-layer polyelectrolyte nanocoating43, and that individual cells, including Bacillus spores, can then be subsequently encapsulated with silica44; the silica encapsulation greatly enhanced viability by protecting the cell from harsh environments.

By 1999 and thereafter, DOD (Department of Defense) interest in pathogen encapsulation was explicitly spelled out in (unclassified) budget documents for the Biological Warfare Defense program at DARPA (the Defense Advanced Research Projects Agency) and the Chemical/Biological Defense program, which includes Dugway.

DARPA’s Biological Warfare Defense program was conducting a project, initiated in 1995, to develop a miniature time-of-flight mass spectrometer for rapid detection of a broad spectrum of chemical and biological warfare agents in aerosol form45. Johns Hopkins University’s Applied Physics Laboratory (APL) took the lead, with critical collaboration from USAMRIID to provide and test pathogens and develop a database of their mass spectral signatures; this work included preparation of both Sterne46 and Ames (inactivated) Bacillus anthracis powders – the latter grown from an RMR 1012 inoculum in late 200047. Dr. John Ezzell of USAMRIID, who acted as the FBI’s scientific advisor on anthrax, speaking from the floor at a seminar on the Amerithrax investigation on Nov. 29, 2010, described his production of (sterile) anthrax spores at that time, commenting that the powders produced in his lab were purer than any of the letter powders48 and that high purity was needed for determining unique mass spectral signatures49,50.

From 1999 to 2001, annual DOD budget item justifications for the DARPA project on detectors (called “sensors”) listed a plan to be carried out in FY 2001 to “evaluate methods for removing microencapsulation of disguised pathogens and/or sensing through the micro-encapsulation51”. The budget item justification sheet dated February 200252 listed that plan under “FY2001 Accomplishments”, indicating that at least one microencapsulated pathogen was studied by DARPA in 2001.

Also accomplished by DARPA in 2001 was the evaluation of “methods of cell stabilization for possible application to cell based sensors”. One such stabilization method is microencapsulation.

Concomitantly, the Chemical/Biological Defense Program (CBDP)53, covering work at Dugway, undertook in 1999 to identify and evaluate emerging threat agents by various means, and continued work on this project through FY2001, during which year they assessed the gaps in the threat agent data and the needs for improved simulants. Also in 2001, plans were made to initiate a program of synthesis, toxicology screening and characterization of new threat materials, to include Fourth Generation Agents [which include those altered for better survival54, e.g. by microencapsulation], and to initiate development of improved simulants for microencapsulated viruses and stabilized bacteria. Throughout this period the program provided controlled biosimulant aerosol challenges for Joint Service, DARPA, and DOE experimental equipment. Dugway tested DARPA’s detection equipment in 199955.

Because of Dugway’s emphasis on improved simulants and on stabilization/survivability, Dugway may be the source of the unique B. subtilis contaminant found in the early attack letters56. There is no evidence that Dugway or any other site was ever examined for the presence of the critical Bacillus subtilis strain. The strain was found to be a hypersporulator57, typical of the strains that originated at Dugway for use as simulants58. The Bacillus subtilis isolated from the NY Post letter was sequenced for the FBI59 and found to have greater than 98% identity60 with the widely-studied strain Bacillus subtilis 168 (sequenced in 199761), often used as a surrogate for Bacillus anthracis62 and as the standard model in studies of the molecular and genetic basis of Bacillus spore resistance to environmental stresses63. Research on Bacillus genetics was proliferating at the time of the anthrax attacks.

Dugway is the only place known to have made live, dry, weaponsgrade anthrax powder in the years before the attacks64. During the Amerithrax investigation the Dugway laboratory was the place the FBI asked to conduct experiments attempting to reverse-engineer production of the attack anthrax powders. The Dugway laboratory had supplied USAMRIID with most of the spores in flask RMR 1029, the putative parental source of the attack anthrax, in 1997.

A B. anthracis stock sample provided by Dugway to the FBI Ames anthrax repository tested positive in at least one of the four genetic assays used as indicators of relationship to the attack anthrax65, and the NAS committee believed that Dugway probably produced all four of the genetic markers used for assays66. A subcontractor67 working at Dugway in 2001, Battelle Memorial Institute, had twice received material from RMR 1029 from USAMRIID during 200168, upon receipt of which Battelle was given permission “to provide aliquots to other laboratory facilities for legitimate research purposes69”.

Battelle is well-known for aerosol expertise, and was working with dry anthrax simulant spores in the period before the attacks70. It would probably be difficult to distinguish whether Dugway or Battelle personnel were responsible for any anthrax-related work done at Dugway at that time.

The elemental composition of the 2001 attack anthrax presents critical clues that were not considered or were misinterpreted throughout the original investigation. Extensive experimental data released by the FBI after the anthrax case was closed make it possible to trace some of the implications of these clues: the substantial presence of tin, a toxic material that must have been added subsequent to growth, and a uniquely high content of silicon in the attack spores. No Bacillus spore preparations other than the attack anthrax have ever been found to contain such a high level of silicon, although some surrogate spore powders prepared at Dugway following FBI instructions have been cited as evidence that high levels of silicon can be reproduced; however, examination of the experimental data reveals that the silicon in these samples was unquestionably an artifact. The elemental evidence suggests that the attack spores had been coated with silicone (a polysiloxane) in the presence of tin, which catalyzes the cross-linking of polysiloxane chains needed to form an encapsulating coating on the spore coat. Microencapsulation helps protect biological agents from damage during atmospheric exposure and from the body’s defenses during infection, and would defeat some detection methods. Microencapsulation, which would explain the location and amounts of both tin and silicon in the attack spores, requires special expertise and sophisticated facilities. DOD-sponsored projects explicitly involving microencapsulation at DARPA, Dugway and perhaps elsewhere were spelled out publicly in budget documents in 1999 and thereafter, and executed at the very time of the anthrax attacks. Both the Dugway laboratory and Battelle Memorial Institute, a sub-contractor at Dugway, had extensive experience in making Bacillus spore powders; both had access to Bacillus anthracis genetically matching the attack spores; both could have made the attack spores legally for institutions conducting biodefense activities that required microencapsulated spores. Furthermore, a small but significant amount of tin, about 4% of that in the attack spores, has been found in some surrogate spore products made at Dugway. A measureable tin content has not been found in any other Bacillus spores except the attack spores. The tin in the Dugway surrogates may have been a remnant, indicative of earlier, classified work. Avoidance of governmentsponsored, classified research may account for some of the limitations of the investigation.

Introduction

The FBI’s investigation of the 2001 anthrax attacks focused heavily on genetic evidence that strongly links the attack anthrax to a liquid suspension of Ames-strain Bacillus anthracis spores in a flask known as RMR 1029, found at USAMRIID (the US Army Medical Research Institute for Infectious Diseases). Prior to the attacks, spores from this flask − believed to be the parental source of the anthrax in the letters – had been sent to a number of other laboratories. Collection and analysis of an FBI repository of samples from all laboratories known to possess the Ames strain indicated that several laboratories had Bacillus anthracis that was identical or nearly identical to that in RMR 1029. There is no public information, however, as to whether any of those laboratories may have produced the attack anthrax, possibly as part of their authorized work1.

In addition to the genetic composition of the attack anthrax, the elemental compositions of the spore powders provide equally important clues: the presence of tin, a toxic material that must have been added subsequent to growth; and a uniquely high content of silicon, not in the familiar form of silica nanoparticles. Examination of the extensive (but incomplete) experimental data released by the FBI in February 2011 and of other evidence from government sources has made it possible to trace some of the implications of these clues2. It is remarkable that there is no evidence that the FBI tried to determine the chemical forms of the tin and silicon in the attack powders, although that could have been (and still could be) done straightforwardly by spectroscopic methods. Perhaps the FBI knows the answers but has concealed them in order to keep potentially dangerous information out of the hands of adversaries.

Significance of tin in the attack anthrax

Analysis by the FBI for tin in the attack samples, using ICP-OES (inductively-coupled plasma optical emission spectrometry), found 0.1979 wt% tin in the Leahy powder and 0.6511 wt% in the NY Post sample3. In contrast, many other Bacillus spore preparations4 from various sources that were subjected to elemental analysis by the FBI or its contractors were found to contain no tin at all (see data in Table 1, and summary of tin contents in Table 2).

Table 1: Tin and Silicon in Bacillus Spores.

Table 2: SummaryA: Tin in Bacillus Spore Preparations.

To explain the substantial presence of tin in the attack spores, which has never been addressed by the FBI, Hugh-Jones et al.5 have proposed that the spores were processed after growth by coating them with silicone, typically a polysiloxane formed by hydrolysis and polymerization of a silane compound, in the presence of tin. Tin catalyzes the cross-linking of polysiloxane chains, which would thereby form an encapsulating silicone coating on the spore coats6 – the location at which tin, and silicon as well, have been found7 in the attack spores. This process, which would explain the presence, location and amounts of both tin and silicon, would require special expertise and sophisticated facilities. It is an aim of this paper to explore the evidence that the spores used in the letter attacks may have been microencapsulated for legitimate biodefensive purposes before they fell into the hands of the letter sender(s).

“Reverse engineering” of the silicon content of the attack spores

Natural incorporation of silicon by Bacilli, from silicates in growth media, was first reported in 19808. Sandia National Laboratory has shown that the silicon in samples made at that time, as well as Bacillus samples more recently produced in other laboratories, is located at the spore coat9. All of the many Bacillus anthracis and other Bacillus spore preparations with naturally-incorporated silicon that have been analyzed by the FBI or its contractors have contained a maximum of 0.5 wt% silicon, usually much less, often zero (Table 1), with the sole exception of eight samples from a set of 36 agar-grown Bacillus anthracis spore powders prepared in 2003 at Dugway (DPG, Dugway Proving Ground) according to FBI instructions, in an effort to “reverse engineer” the letter powders (data in Table 1, silicon contents summarized in Table 3). The 36 samples comprised all permutations of: two media, two washing procedures, four drying procedures, and three milling procedures10. Based on the plan and the extensive experimental data provided by Dugway11, there appears to have been no intentional effort to reproduce the silicon or tin in the attack spores; the emphasis seems to have been on particle size and viability. However, at that time there was considerable public concern about the silicon in the attack spores (the presence of tin had not been divulged by the FBI).

Table 3: SummaryA: Silicon in Bacillus Spore Preparations.

The FBI selected only ten of these “surrogate” samples for elemental analysis, using ICP-OES in an FBI laboratory. Eight of the ten samples were found to contain unusually high levels of silicon (0.5-5 wt%, average 1.4 wt%)12. These eight are the only known Bacillus preparations, other than the attack spores, containing more than 0.5 wt% silicon. These eight samples were all ball-milled, while the remaining two samples, which contained only 0.2 and 0.3 wt% silicon, were milled by hand in a porcelain mortar and pestle13,14. The FBI did not choose to examine any of the samples that had been milled by a third procedure – a manual stainless steel ball and sieve method.

The ball-milled samples also contained extremely high levels of aluminum (0.4-3 wt%), while the mortar-and-pestle-milled samples contained only 0.0087 and 0.0149 wt% aluminum15 (The NY Post and Leahy samples contained 0.0158 and 0.0220 wt% aluminum, respectively).

The ball mill described by Dugway consisted of a porcelain milling jar containing a grinding medium consisting of zirconium cylinders, rotated for approximately 16 hours. Porcelain is composed of silica and alumina. The presence of a high level of aluminum only in the ballmilled samples is telltale and would be hard to miss. Furthermore, the Dugway laboratory noted that all ball-milled samples (but not those milled by other methods) showed a high degree of clumping, as viewed microscopically, making spore viability determination impossible16, and the FBI laboratory noted that the Dugway samples were abnormal in not fully responding to the usual acid digestion procedure, as a consequence of which the concentrations of two of the samples in particular-those with the highest silicon content (5 and 2 wt%) – were likely inflated17. These observations signal that most or all of the silicon and aluminum in the eight ball-milled samples was an extra-sporular artifact. Analyses done at Sandia of two of the ball-milled Dugway preparations (those containing 5 and 0.7% wt% silicon, NDLB and SDLB in Table 1), using STEM-EDX (scanning electron microscopy with energy-dispersive X-ray analysis) measurements on thin sections, confirm that none of the silicon was in the spore coat18, where it is located both in the attack spores and in Bacilli containing naturally-incorporated silicon19.

The question arises as to why the FBI wanted any of the Dugway samples to be ball-milled, or milled at all, given that the purpose of making the samples was “to give some insight into how the materials in the [anthrax] letters were made20”, and given that the Dugway samples were made in 2003, well after Battelle (Battelle Memorial Institute, BMI, a defense contractor) had prepared unmilled Bacillus globigii surrogates that compared favorably to the Leahy attack spores with regard to dispersibility, as indicated by aerosol particle size distribution (see next section). Battelle’s report to the FBI on particle sizes, dated February 2002, noted that their result “indicates that neither milling nor other processing of a freeze-dried B. anthracis spore powder was required21”.

The FBI/Dugway sample preparation plan22 had called for some additional samples to be dried by an (undescribed) acetone method in use at Dugway, but this part of the plan was evidently set aside, although portions of pastes from two other samples were eventually acetonedried23 – but they were never named, further processed, described or analyzed, at least not in publicly-released documents. Acetone drying might have obviated any reason for milling: Bruce Ivins, who assisted the FBI in the early part of the investigation, later quoted one of his USAMRIID colleagues as telling him “he’d use an organic solvent like acetone or alcohol to pull water out of purified spores and then easily make them into powder24”.

Along with the 36 agar-grown samples, Dugway had also prepared two surrogate samples by fermentation in liquid Leighton- Doi medium25. This was an interesting experiment, in which one fermentation sample was grown in the presence of an antifoam agent containing polydimethylsiloxane (Sigma Antifoam C), while the other was grown with an antifoam containing no silicon (Sigma Antifoam 204). Did the spores that had been exposed to polydimethylsiloxane during growth contain a higher level of silicon than has been found in spores exposed only to silicates in media? If so, might the attack spores have been exposed to polydimethylsiloxane? The only relevant information released to the NAS Committee and the public was Sandia’s observation that a Dugway fermentation sample received from the FBI under the code name “040255-1,” for determination of silicon in the spore coats, contained some silicon on about 25% of the spore coats26. This information was used solely as evidence that some fermentation samples contain some silicon. Which of the two fermentation samples this was, and how much silicon was on the spore coats and in the bulk sample, were not revealed to the NAS or the public. Sandia noted, however, that, although some of the various surrogate samples they studied contained silicon on the spore coat, compared to the attack samples “the details are different…chemistry, distribution27”.

The silicon content of the eight ball-milled Dugway agar-grown surrogates has been wrongly cited as evidence that high levels of silicon can be reproduced in Bacillus anthracis preparations28. Similarly, the fact that small amounts of silicon can sometimes be incorporated naturally into the coats of Bacillus spores during their growth has been cited to imply that the much larger amounts of silicon in the attack spore powders must result from the same process29, even though no growth experiments have actually reproduced the high silicon content, nor has the chemical nature of the silicon in the two cases been determined and compared. This selective use of partial information may have convinced many who have not looked into the experimental details that the silicon question has been resolved.

Dispersibility of the attack anthrax spores as aerosols

Because of the suspicion that the silicon in the attack spores might have been there to increase their aerosol dispersibility, Battelle – known for its aerosol expertise – was asked to compare the aerosol particle size distributions of the attack spores to those of dry simulant spore preparations made at Battelle without any special post-growth processing30. The simulants were two Bacillus globigii powders, “washed” and “unwashed,” isolated only by centrifugation, water-washing (or not) and lyophilization31. The aerosol particle size distributions of the Bacillus globigii samples were found to be bimodal, with 1-2% in the single spore range; their distributions were similar to, and slightly narrower than, that of a sample taken directly from the Leahy letter and analyzed in February 200232. The similarity of the results for the Leahy and surrogate samples appears to confirm the FBI’s repeated insistence that the attack spores had not been treated in any way that affected aerosol dispersibility. These results can best be understood in light of the observations of Dr. Thomas Geisbert at USAMRIID, who measured particle sizes in the pristine Daschle sample by electron microscopy and reported that hydrated samples consisted of single spores – which accounts for the Daschle titer of 2.1×1012 cfu/g, the theoretical limit for pure, 100% viable, single Ames spores; but in dry samples (such as those used for aerosol studies) Geisbert found that the spores aggregated to form clumps of up to 105 spores33.

Battelle initially (October 2001) examined the aerosol particle size distributions of two Daschle powders provided by the FBI right after the attack, labeled SPS.57.0334 and SPS.57.0835, neither of which was taken directly from the attack letter itself36. These were the results that were most frequently cited by the FBI. Both the Daschle samples had very low titers (4.6×1010 cfu/g and 1.5×108 cfu/g, respectively) and showed other signs of contamination and aggregation37. It is therefore not surprising that the particle size results for these samples compare poorly with the Bacillus globigii results. Why did the FBI choose to send samples recovered from spills in Daschle’s office, rather than a pristine sample, for this important early test? Only the Leahy data, obtained some four months later and largely ignored, can be considered significant, allowing the tentative conclusion that the attack spores probably had no special advantage or disadvantage for aerosol dispersion.

Microencapsulation of pathogens in US biodefense research

Although there is no evidence to indicate that the tin and silicon content of the spores conferred any benefit for purposes of the letter attacks, their presence is meaningful if the attack spores had been prepared legitimately for other purposes. Silicone microencapsulation would have been desirable for increasing the resistance of the spores to inactivation by hazards such as UV light, ozone or toxic materials38, and for preventing detection of the spores by some methods. (It is at the spore coat, rather than the external membrane (the exosporium) of Bacillus anthracis that these functions occur39). These are properties of military concern. The use of microencapsulation for such purposes was already an old idea40 in the biodefense community in the years immediately preceding the attacks. Microencapsulation by special polymers to produce particles in the 1-10 micron range could protect microbes from environmental damage during aerosolization and delivery [e.g. via bomblets] and also from the body’s initial defenses during the infection process, according to an encyclopedia on weapons of mass destruction published in 2004, and could also help defeat some detection schemes41. The encyclopedia notes that the technology requires an advanced research and development infrastructure, unlikely to be available to terrorists, but “state-level CBW programs could certainly employ microencapsulation to produce highly effective weapons of mass destruction42”.

In the non-military literature it has recently been shown that single, living Bacillus spores can be encapsulated using layer-by-layer polyelectrolyte nanocoating43, and that individual cells, including Bacillus spores, can then be subsequently encapsulated with silica44; the silica encapsulation greatly enhanced viability by protecting the cell from harsh environments.

By 1999 and thereafter, DOD (Department of Defense) interest in pathogen encapsulation was explicitly spelled out in (unclassified) budget documents for the Biological Warfare Defense program at DARPA (the Defense Advanced Research Projects Agency) and the Chemical/Biological Defense program, which includes Dugway.

DARPA’s Biological Warfare Defense program was conducting a project, initiated in 1995, to develop a miniature time-of-flight mass spectrometer for rapid detection of a broad spectrum of chemical and biological warfare agents in aerosol form45. Johns Hopkins University’s Applied Physics Laboratory (APL) took the lead, with critical collaboration from USAMRIID to provide and test pathogens and develop a database of their mass spectral signatures; this work included preparation of both Sterne46 and Ames (inactivated) Bacillus anthracis powders – the latter grown from an RMR 1012 inoculum in late 200047. Dr. John Ezzell of USAMRIID, who acted as the FBI’s scientific advisor on anthrax, speaking from the floor at a seminar on the Amerithrax investigation on Nov. 29, 2010, described his production of (sterile) anthrax spores at that time, commenting that the powders produced in his lab were purer than any of the letter powders48 and that high purity was needed for determining unique mass spectral signatures49,50.

From 1999 to 2001, annual DOD budget item justifications for the DARPA project on detectors (called “sensors”) listed a plan to be carried out in FY 2001 to “evaluate methods for removing microencapsulation of disguised pathogens and/or sensing through the micro-encapsulation51”. The budget item justification sheet dated February 200252 listed that plan under “FY2001 Accomplishments”, indicating that at least one microencapsulated pathogen was studied by DARPA in 2001.

Also accomplished by DARPA in 2001 was the evaluation of “methods of cell stabilization for possible application to cell based sensors”. One such stabilization method is microencapsulation.

Concomitantly, the Chemical/Biological Defense Program (CBDP)53, covering work at Dugway, undertook in 1999 to identify and evaluate emerging threat agents by various means, and continued work on this project through FY2001, during which year they assessed the gaps in the threat agent data and the needs for improved simulants. Also in 2001, plans were made to initiate a program of synthesis, toxicology screening and characterization of new threat materials, to include Fourth Generation Agents [which include those altered for better survival54, e.g. by microencapsulation], and to initiate development of improved simulants for microencapsulated viruses and stabilized bacteria. Throughout this period the program provided controlled biosimulant aerosol challenges for Joint Service, DARPA, and DOE experimental equipment. Dugway tested DARPA’s detection equipment in 199955.

Because of Dugway’s emphasis on improved simulants and on stabilization/survivability, Dugway may be the source of the unique B. subtilis contaminant found in the early attack letters56. There is no evidence that Dugway or any other site was ever examined for the presence of the critical Bacillus subtilis strain. The strain was found to be a hypersporulator57, typical of the strains that originated at Dugway for use as simulants58. The Bacillus subtilis isolated from the NY Post letter was sequenced for the FBI59 and found to have greater than 98% identity60 with the widely-studied strain Bacillus subtilis 168 (sequenced in 199761), often used as a surrogate for Bacillus anthracis62 and as the standard model in studies of the molecular and genetic basis of Bacillus spore resistance to environmental stresses63. Research on Bacillus genetics was proliferating at the time of the anthrax attacks.

Dugway is the only place known to have made live, dry, weaponsgrade anthrax powder in the years before the attacks64. During the Amerithrax investigation the Dugway laboratory was the place the FBI asked to conduct experiments attempting to reverse-engineer production of the attack anthrax powders. The Dugway laboratory had supplied USAMRIID with most of the spores in flask RMR 1029, the putative parental source of the attack anthrax, in 1997.

A B. anthracis stock sample provided by Dugway to the FBI Ames anthrax repository tested positive in at least one of the four genetic assays used as indicators of relationship to the attack anthrax65, and the NAS committee believed that Dugway probably produced all four of the genetic markers used for assays66. A subcontractor67 working at Dugway in 2001, Battelle Memorial Institute, had twice received material from RMR 1029 from USAMRIID during 200168, upon receipt of which Battelle was given permission “to provide aliquots to other laboratory facilities for legitimate research purposes69”.

Battelle is well-known for aerosol expertise, and was working with dry anthrax simulant spores in the period before the attacks70. It would probably be difficult to distinguish whether Dugway or Battelle personnel were responsible for any anthrax-related work done at Dugway at that time.

A new clue

FBI documents released in 2011 show that the ten “reverse engineered” surrogate samples made at Dugway for the FBI contained a small but significant amount of tin. The FBI has never commented on this finding, nor on the tin content of the attack spores, and there is no evidence that the “reverse engineering” experiments71 or any other investigations were aimed at reproducing or explaining the presence of tin. No other Bacillus spore preparations except the attack spores contained tin (Table 2). The tin content of the Dugway samples, measured by ICP-OES in the FBI laboratory72, varied from 0.0033- 0.0266 wt% (average 0.0086 wt% tin). Omitting the two samples for which the FBI laboratory reported that the analytical results were most questionable73 gives a range of 0.0033-0.0084 (average 0.0058 wt%). In any case, the Dugway surrogates contained about 3-4% as much tin as was found in the Leahy spores (0.197 wt%) (data in Table 1). A “commercially available multi-element standard solution74” analyzed at the same time had 0.0069 wt% (69 ppm) tin75. The presence of tin cannot be attributed to ball milling; the two samples that were not ballmilled contained 0.0038 and 0.0064 wt% tin, consistent with the tin in the eight ball-milled samples. No tin was found in any culture medium components76 nor was tin exposure likely during growth or in any of the post-growth treatments77.

Random, trace contaminants are extremely unlikely to be found by ICP-OES in the concentration range observed in the Dugway samples. Analyses of a set of Bacillus thuringiensis samples at Sandia National Laboratory using TOF-SIMS (time of flight secondary ion mass spectrometry), a more sensitive method than ICP-OES78, found traces of tin, too small to quantify, in those samples79. One of the Sandia authors (J. Michael) is quoted80 as saying “we were surprised at first [at the traces of foreign elements], then we realized that the elements could have come from any number of sources − lab equipment, a residual cleaning solution, some other kind of contamination”. Contaminants that were not quantifiable by TOF-SIMS would certainly not be observable or quantifiable by ICP-OES81.

The small but real tin content found by the FBI laboratory in the Dugway samples appears to have been overlooked. Was it a remnant of tin used previously at Dugway in classified work? There is no evidence whatsoever to rule out the possibility that the attack samples had been legally made at Dugway82 or elsewhere. It must be recognized, of course, that making the spores is not synonymous with sending the letters. The “attack” anthrax could have been made for the use of US agencies or contractors conducting legitimate activities such as vulnerability and response assessment or testing detection devices such as DARPA’s. The letter sender(s) may have been one or more individuals who, whether legally or illegally, had access to the material at some point in the process.

Discussion

Tin, found by the FBI in substantial amounts on the spore coats of the attack anthrax, has not been discussed or investigated. Tin is toxic to bacteria and therefore must have been acquired by the attack spores after their growth. The quantities are too high to be accidental contaminants. In approximately the amounts found83, tin is known to be a catalyst in the formation of silicone coatings for microencapsulation. Although there may be other possible explanations for the presence of tin, neither the FBI nor anyone else has put forward an alternative. The microencapsulation hypothesis is strengthened by the evidence that government-sponsored programs specifically involving microencapsulated pathogens were in progress at the very time of the attacks.

Silicon, in uniquely high amount, was also found on the coats of the attack spores. Although the FBI reported similarly high silicon content in a set of surrogate Bacillus anthracis spore powder preparations “reverse engineered” at Dugway, examination of the laboratory data (released after the case was closed) reveals that the silicon present was unquestionably a milling artifact and had no connection to the spores. Neither the Dugway surrogate spores nor any of the many other Bacillus spore preparations examined have ever been found to contain levels of silicon comparable to the attack spores (Table 3). These facts cast strong doubt on the FBI’s explanation that the silicon in the attack spores was naturally incorporated during growth. The FBI’s investigation of the silicon issue has been characterized by loose assumptions and sketchy data, raising fundamental questions about the investigation’s approach to the whole matter of additives in the attack spores.

The presence, shown by FBI analysis, of the two extraneous elements, tin and silicon, together in the attack spores favors the silicone microencapsulation hypothesis. Microencapsulation, a process that would require special expertise and sophisticated facilities, could explain the presence, location and amounts of both elements. At least two government programs, at DARPA and Dugway, had projects requiring microencapsulated pathogens or simulants. Both Dugway and Battelle, a sub-contractor there, had access to Bacillus anthracis from the presumptive parental flask RMR 1029. Both had the expertise to make anthrax spore powders, both – and perhaps other government- supported laboratories as well – could have made the attack spores legally for institutions conducting biodefense activities that required microencapsulated spores.

Other than the attack spores, no Bacillus preparations have ever been found to contain tin, with one exception: the set of ten Bacillus anthracis powders “reverse engineered” at Dugway in 2003 (Tables 1 and 3). These samples were produced by standard, well-described preparation procedures, with no known exposure to tin. Nonetheless, FBI analyses that became available after the case closed found a small but real amount of tin in the Dugway powders, about 4% of the amount in the attack spores, too large to be attributed to random, trace contamination; contamination from a previous use of tin appears to be the most likely explanation. The tin in the Dugway samples may be an indicator of previous, classified work with tin at Dugway to provide materials for biodefense activities.

Many US agencies were involved in biological antiterrorism activities at the time of the anthrax attacks. The Chemical and Biological Defense Program initiated in FY 2000 a “broad CB countermeasures program to enhance ability to recognize, prevent, respond to, mitigate, and recover from a CB terrorist incident84”. There had already been an epidemic of hoax anthrax letters. CIA scientists possessed Ames anthrax and were working with other government agencies and outside contractors [including Battelle85] in the defensive biowarfare program86; former biological weaponeer Bill Patrick wrote a classified paper about anthrax sent by mail, dated February 1999, for SAIC (Science Applications International Corporation) under CIA contract87. Nonetheless, the CIA issued a statement that they were unaware of any project to assess the impact of anthrax sent through the mail88; similarly, the FBI claimed to have been blindsided by a letter attack89.

The evidence indicates that live, microencapsulated Bacillus anthracis spore powders probably existed legitimately at the time of the attacks and were the likely sources of the anthrax in the attack letters. A number of individuals would have had access to such materials, whether legally or illegally. For the letter-sender(s), the presence of additives in the powders would have been irrelevant. For the FBI, a real investigation of the presence of additives may have been impossible without ‘off-limits’ intrusion into classified biodefense matters90.

2Sets of FBI Documents identified by B (Batch) and M (Module) numbers were provided by the FBI to the National Academy of Sciences (NAS) Committee on the Review of the Scientific Approaches Used During the FBI’s Investigation of the 2001 Bacillus anthracis Mailings Investigation; the documents are listed in the NAS Report issued February 15, 2011, pp. 133ff, Index of Documents Provided by the Federal Bureau of Investigation; the documents were released to the public in February 2011 and were available from the NAS at that time.

4The only exception, a set of 10 “reverse engineered” samples made at Dugway, is discussed in a later section.

5Hugh-Jones et al., op. cit.

6See Hugh-Jones et al. (op. cit.) for details of the proposed siliconization procedure, which requires moisture from within the spores and results in deposition on the spore coat, not on the exterior surface of the spore (the exosporium).

13The milling methods used by Dugway for the 36 samples are described in FBI Document B1M13, p. 71.

14The mortar-and-pestle- milled samples, labeled NDLM (0.3 wt% silicon) and NPLM (0.2 wt%), can be compared to their ball-milled counterparts differing only in milling method: NDLB (5 wt% silicon) and NPLB (0.5 wt%). Note that NDLB was one of the two samples for which the analytical results were questionable, according to the FBI laboratory report (B1M7, p. 28); the other was SPVB (2 wt%). These two are the samples with the highest analytical results for silicon.

15FBI Document B1M7, FBI Elemental Analysis Summary, p. 93.

16FBI Document B1M13 (Dugway), pp. 23 and 84-85, where it can be seen that all 12 of the ball-milled Dugway samples, not just those analyzed by the FBI, exhibited clumping.

17FBI Document B1M7 (FBI Laboratories), p. 28.

18NAS Report “Review of the Scientific Approaches used during the FBI’s Investigation of the 2001 Anthrax Letters,” Feb. 15, 2011, p. 67 (analysis) and p. 70 (identity of the samples studied).

19Michael J, Kotula P (2009), op. cit.; also Sandia reports to the FBI in FBI documents B1M6 and B1M1 (in these reports, samples are coded and it is sometimes difficult or impossible to find their identities).

20FBI Document B1M13 (Dugway), p. 93.

21FBI Document B2M13 (Battelle), p. 151.

22FBI Document B1M13 (Dugway), Test Plan, p. 15.

23FBI Document B1M13 (Dugway), p. 79.

24Amerithrax Investigative Summary, February 19, 2010, p. 75.

25B1M13 (Dugway), pp. 70 and 74-5. The two fermentation products, grown in 2003 at the same time and from the same stock as the agar samples, were stored until 2005, when they were irradiated and processed (methods not disclosed) under the designations “Lot 05AUG05” for one grown with Antifoam 204 and “Lot 01SEP05” for one grown with Antifoam C.

26FBI document B1M1(Technical Review Panels), Sandia report on elemental mapping of Amerithrax Samples, pp. 109, 110, 100, 106. The data for this sample, coded 040255-1, in Sandia’s Table on page 109 are the same as those for the “Dugway surrogate (fermentation using Leighton-Doi media)” in the NAS Report “Review of the Scientific Approaches Used During the FBI’s Investigation of the 2001 Anthrax Letters,” Feb. 15, 2011, pp 67 and 70, indicating that the FBI must have identified the coded material to the NAS Committee as a Dugway fermentation sample, but evidently did not specify which one.

31FBI Document B2M13 (Chemical and Physical Characteristics, Battelle), The Analysis of Surrogate Dry Powder Bacillus Spore Product, pp. 92ff. The “unwashed” preparation had a titer of 9×1011 cfu/g, an indication of its high quality (compare to the titer of the pristine Daschle sample, 2.1×1012, which had been determined at USAMRIID just after the material was received from the Capitol Police (FBI Document B1M2 (USAMRIID), Anaytical Test Report, pp. 36ff; note pp. 37 and 42). The titer of spores in the Leahy letter has never been reported.

32FBI Document B2M13 (Chemical and Physical Characteristics, Battelle), Summary of Sample Analyses, pp. 146ff. Analysis of the Leahy sample was done in February 2002, after the Leahy letter had been stored in a mail bag for a number of months; it is therefore possible that the Leahy spores were no longer in their original condition.

47On August 28, 2000, forty ml were removed from flask RMR 1029 at USAMRIID “for DARPA mass spec project with JHU-APL” (inventory control sheet, http://www.vault.fbi.gov/ amerithrax, part 24, p. 8); see also, FBI interview believed to be of Dr. Joany Jackman, a major participant in the DARPA project working at USAMRIID under John Ezzell from 1997-2000 and then at Johns Hopkins APL: B. anthracis, grown from an inoculum (about 1012 cfu/ml) provided by Bruce Ivins and purified on a gradient, was used at USAMRIID for the aerosol work (http://vault.fbi.gov/Amerithrax/ part 21, pp 19-22; Jackman’s corroborating biographical details are at http://www.hopkinsmedicine.org/ medicine/std/team/Jackman.html).

64New York Times, US Recently Produced Anthrax in a Highly Lethal Powder Form, December 13, 2001.

65FBI Document B2M10 (Statistical Analysis), especially Appendix V of the Report on Statistical Analysis, where the names of some specific laboratories that submitted repository samples of interest are handwritten next to the sample data: DPG (Dugway Proving Ground); BMI (Battelle Memorial Institute); DRES (Defense Research Establishment, Suffield, Canada); NMRC (Naval Medical Research Center); USAMRIID. See also the National Academy of Sciences (NAS) Report “Review of the Scientific Approaches used during the FBI’s Investigation of the 2001 Anthrax Letters,” February 15, 2011, pp. 110-112, and FBI Document B2M10, p. 25. From information in these documents it appears that seven repository samples from USAMRIID and one from Battelle tested positive in all four genetic assays; one sample each from USAMRIID and Northern Arizona University (an FBI contractor in the anthrax case) had three positives; a samples from DRES had two positives; and samples from six other laboratories, including Dugway and the Naval Medical Research Center plus four unnamed laboratories, tested positive in one or two assays, as did additional samples from some of the laboratories already mentioned.

66NAS Report (op. cit.), p. 108 gives reasons why all four markers probably originated at Dugway; the Report also discusses the probability of false negatives and presents a Table of assay results on 30 repeat samplings of flask RMR 1029 (the putative parental source of the attack anthrax) as an illustration (p. 117).

67Personal communication January 11, 2002 from David Lore, Columbus Dispatch Science Reporter, author of article “Labs deny use of letter anthrax: Powdered spores not part of stocks, Battelle official says” in Columbus Dispatch, January 9, 2002.

68FBI Document B3D16. One Ames repository sample from Battelle tested positive in all 4 assays; another from Battelle, known to have originated from an RMR 1029 sample, tested positive in 2 of 3 assays (FBI Document B2M10, p. 25).

69Maureen Stevens et al. vs United States of America: Notice of Errata, Document 162, entered on FLSD Docket 07/19/2011, submitted by the Defendant United States in US District Court, Southern District of Florida, Case Number: 03-81110-CIV-Hurley/Hopkins.

70Columbus Dispatch, Labs deny use of letter anthrax: Powdered spores not part of stocks, Battelle official says, January 9, 2002.

71See FBI/Dugway plan for the reverse engineering work in FBI Document B1M13.

72Elemental analyses of the 10 samples, measured by ICP-OES in an FBI laboratory, are given in FBI Document B1M7 (FBI Laboratory Reports), Samples from Dugway, pp. 26-28; the data are repeated at B1M7, Elemental Analysis Summary, p. 93.

75FBI Document B1M1 (Technical Review Panels), p. 83, gives the elemental analysis of the Standard, together with averages for the elements in the ten Dugway samples (presented at a Chemistry Review Panel in August 2005).

80ScienceInsider, New Challenge to FBI’s Anthrax Investigation Lends an Ear to Tin, 11 October 2011.

81For sensitivities of the analytical methods see Table 1.

82The finding, via stable isotope analysis (B1M9, p.44), that Dugway water is unlikely to have been used to grow the attack spores is probably not relevant; Dugway’s report (B1M13) on preparation of the surrogate samples mentions the use of “sterile water for irrigation” and “sterile water for injection,” which are generally purchased in small bottles from distant providers. The FBI laboratory, in analysing media components (B1M7), included “Baxter sterile water for irrigation”.

90An appropriately targetted investigation would seek to determine whether Bacillus anthracis had been microencapsulated prior to the letter attacks, and, if so, by whom, where, when, and the amounts, strains and dispositions of the resulting materials. Parts of such an investigation might still need to be classified.

DXersaid

The pictures on the vials confirm it was not the handwriting of Dr. Bruce Ivins. I will upload them as the weather permits. Dr. Fellows worked in Building 1412. CDC 7738, which was genetically matching, was located in Building 1412. Why did the FBI on or about August 8, 2008 claim that the genetically matching virulent Ames was only in Building 1425, when it knew that it was in Building 1412? That was reckless.

All the four morph analysis did was to winnow those with access from 700-1000 (depending on the estimate) to up to 300. To cast it as a smoking gun is baseless. Moreover, US Attorney Taylor made it seem that the genetically matching Ames was only stored in Building 1425 — and suggested that only 100 needed to be eliminated. Instead, up to 300 needed to be eliminated. And that was just at USAMRIID — and anyone at USAMRIID could have transferred the genetically matching Ames to anyone!

In particular, why didn’t the FBI disclose the fact of the 1998 transfer above? Why wasn’t the contemporaneous correspondence accompanying the transfer produced? The hazardous materials unit was responsible for reviewing the emails and identifying this transfer in connection with the distribution of Ames. (And no you don’t merely assume that scientists in 1998 are complying with EA 101; see civil depositions that I’ve arranged to upload).

Getting the building that the genetically matching Ames was stored wrong was a huge, central and totally unnecessary mistake. Who prepared US Attorney and FBI DC Field Office head Persichini for the briefing?. How can that have been gotten wrong — along with the false claim about the lyophilizer being available to Dr. Ivins. Those were two of the three central points on which US Attorney Taylor’s “Ivins Theory” was based. .

Dr. Decker says he thinks that some receipt was inculpatory. Perhaps he was the one who is the source of the false claim that because the lyophilized was on his hand receipt that it was available to him to use.

Within the hazardous materials unit, who was the origin of the false claim about the lyophilizer being available to Dr. Ivins to use? Why would the receipt be sufficient rather than the real-world location of the lyophilizer?

DXersaid

DXersaid

According to news reports at the time, Louisiana State University and Michigan were subpoenaed in late 2001, long before other labs that were subpoenaed a few months later.

I have written Dr. Hugh-Jones who confirms that the letter above was not to him — he never exchanged photos with Bruce.

Dr. Hugh-Jones/LSU was subpoenaed in late 2001 regarding access to Ames and the B3 there. Based on a news report, I have suggested that the subpoena was limited to only the past two years. Dr. Hamouda, the fellow supplied virulent Ames by Bruce Ivins who was taught by Heba Zawahiri, worked at LSU B3 lab prior to that window. Were documents about his research nonetheless provided to the FBI in 2001? I know the FBI was frequently calling in and there was full cooperation.

He is a leading commentator on the science is exculpatory of Dr. Ivins. His commentatory has prompted repeated editorials in the Washington Post, New York Times and other periodicals calling for Congress to conduct an investigation or the FBI to reopen the investigagiton.

In his patents, Dr. Hamouda thanked genetics expert Kimothy Smith, for the facilities at LSU — along with thanking Hugh-Jones and another scientist who had published Pat Fellows research as a chapter in her PhD thesis. The PF work was featured in the New York Times as potentially making a better bioweapon. As I recall, it involved cloning the virulence plasmids.

Although I have had contact with Dr. Hugh-Jones — most often to compliment him on the lucidity of these articles on the science — I have never felt comfortable asking questions about the research at LSU. I limited myself to having the blog bear down on the question whether the Ames there was matching — and was advised that it was not.

Dr. Hamouda, instead of receiving Ames from LSU (which had acquired it from Peter at Porton Down in the 1990s, Dr. Hamouda was supplied Ames directly by Bruce Ivins (pursuant to the authorized DARPA research).

Years ago I emailed Dr. Hamouda to discuss Dr. Ivins supply of virulent Ames but TH did not respond. His colleague, Michael Hayes, when I spoke to him briefly on the telephone, only said “You don’t want to know.”

Didn’t 7738 get submitted to the repository? In April 2004, the month of Dr. Ivins inquiry to the researcher sent the Ames strain in 1998, Dr. Ivins is corresponding with his assistant about preparing 7736 for submission to the repository. There then was a consent search, I believe, of the lab in July 2004. It was very frustrating for Dr. Ivins not to be told what was taken — making it impossible to know what was what with his inventory. And you can see from the 77 page document uploaded this week by USAMRMC what a detailed and complicated inventory it was. (I can barely keep track of my car keys).

As background, on April 6, 2004, Dr. Ivins discusses this issue of parentage and submission of 7738 to the repository

The numbers were chronological. I believe 7738 would have been created from 7737 (Flask 1029) and not 7739. 7739 would not have been in existence yet.

Thus, couldn’t 7738 be presumed to have been genetically matching and had the four morphs? Couldn’t its parent be assumed to be 7737 (Flask 1029)?

Any Ames prepared from 7738 and sent to an outside researcher can be presumed to be genetically matching to the anthrax mailed in Fall 2001.

“Hi, ____,

Thanks for putting material onto TSA slants from the Bacillus anthracis tube (USAMRIID Agent Registry Number = 2433; USAMRIID Agent Inventory number =7738) for the — repository. I am not sure of the parentage of the strain. Depending upon when the material was made and when it was brought over to ___ it was probably RMR 1029 ____ spores (USAMRIID Agent Registry Number-2432; USAMRIID Agent Inventory number -7737) made in 1997, ___ _______ spores (USAMRIID Agent Registry Number-3502; USAMRIID Agent Inventory number =7739a) made between 1997 and 1999, or ___ ______ spores (USAMRIID Agent Registry Number=3502; USAMRIID Agent Inventory number -7739b and 7739c) made between 1999 and 2001/2002.

Please let know when the entire tube is transferred to the Repository and I’ll take it off the two strain lists.

DXersaid

“IVINS also had samples labeled 7736 and 7738, however, the entire sample has been exhausted. Therefore, he did not provide the FBIR a sample of 7736 and 7738. Sample 7738 was a dilution of 7737.”

7737, remember, was Flask 1029.

So IVINS says that the origin of 7738 was Flask 1029. It was used up or given to someone. The FBI’s analysis of the distribution of Ames was based on self-submission of samples — and was limited to Flask 1029.

The more pertinent question was: did a third party receive Ames from 7738? And if so did it get submitted to the FBIR? And if so, why wouldn’t it have 4 morphs?

Which brings us back to this letter and the unnecessary (b)(6) redactions and the shredding of the civil depositions of the Ivins’ assistants, one of whom would have grown 7738.

What public policy would favor obfuscation of analysis on this question? None. An unredacted copy of the letter should be produced pursuant to a FOIA to some media requestor. (Sandra’s patience is wearing thin with my successive requests given her many other responsibilities).

DXersaid

The following week, in an April 14, 2004 email, Dr. Ivins emphasized in an email to someone:

“Also, Please know that there are many investigators here at USAMRIID that have the Ames strain and have transferred it to other RIID investigators, that __________ was the original holder (at USAMRIID) and transferrer of the strain, and that prior to 2002, it was NOT standard procedure to document every time a RIID investigator gave material to another RIID INVESTIGATOR.”

The email raises the question: Was the email uploaded above supported by a corresponding EA 101 documenting the transfer? An EA 101 would entail a process that confirmed that the receiving facility was authorized to receive the pathogens.

DXersaid

In an April 27, 2004 email, in another email titled “Documents”, Dr. Ivins wrote:

“I don’t recall having informal or unofficial notes saying I received Ames from somebody or gave it to somebody in the Institute. Usually we simply gave strains to fellow investigators working on B. anthracis.”

DXersaid

Dr. Ivins had been responding to an email the previous date on April 26, 2004 that stated:

“”All, please read _________ request below. If you have any records or know of someone else that may have records of intra-lab transfers of Ames spores to 31 DEC 01 please me know ASAP. These records could include lab notes, e-mails or any other log/notes you may have kept. I know that other investigators/visiting scientists may have transferred Ames in the past, if you can recall the names of these individuals please forward them at your earliest convenience.”

DXersaid

By way of some background revealed in an email thread at the time, in an October 12, 2001 email, titled “Ames strain” , Dr. Ivins listed two transfers of Ames in 1998.

He wrote “I, and people in my lab, have sent the so-called “Ames” strain (either original parent or subcultured derivative) of B. anthracis to the following. (By subcultured derivative, I am referring to such strains such as Delta-Ames and ANR, which can be converted to full virulence molecular biology methods, and which would be identified as “Ames” strain.)”

Note: USAMRMC recently uploaded a two-part, 77-page inventory of Ivins strains. USAMRMC has done an absolutely stellar job over the course of years in producing requested documents — once it was freed of the oversight of the meddling DOJ and FBI committee vetting and delaying all productions up until February 2010.

My only wish is that USAMRMC would now consider that DOJ Civil is not making the ubiquitous (b)(6) redactions. The redactions being made by USAMRMC under (b)(6) on balance are not necessary and seriously obstruct analysis.

DXersaid

On October 12, 2001, Dr. Ivins in listing two 1998 transfers of Ames had been responding to an email the same date stating:

“Bruce:

We have a tasker to report to whom we may have sent the Ames strain in the past several years. Do you, or someone else in ___ have any records relating to such information? If so, could you please provide me with that information?

We are checking our MTA’s* as well, but I want to make sure we haven’t overlooked anything.

Thanks for your help!”

*/ A Material transfer agreement (MTA) is a contract that governs the transfer of tangible research materials between two organizations, when the recipient intends to use it for his or her own research purposes. The MTA defines the rights of the provider and the recipient with respect to the materials and any derivatives. Biological materials, such as reagents, cell lines, plasmids, and vectors, are the most frequently transferred materials, but MTAs may also be used for other types of materials, such as chemical compounds and even some types of software.

MTA types[edit]
Three types of MTAs are most common at academic institutions: transfer between academic or research institutions, transfer from academia to industry, and transfer from industry to academia. Each call for different terms and conditions. (Source: Wikipedia)

I have a pending FOIA pending request for a document relating to a FBI Request For Information and all documents in the possession of SJA ever provided to the FBI.

The request also asks for the photographs sent to Bruce of the vials made in the above transfer.

DXersaid

Subject: RE: FW: Strains sent to you in 1998
were all of the vials similar with respect to 1) the type of cryovial; and 2) the handwriting? Sorry to bother you, but I’m just trying to identify the particular parentage of the material we sent you. I don’t need pictures, but if someone could just look at some of the other vials, it would be very helpful. Thanks again!!!

DXersaid

Compare the redacted email to the email forward 10/18/1998 2:37 PM to Ivins containing an internet article entitled “Agent destroys anthrax, doesn’t hurt animals or the environment. Article indicated that Michael Hayes and James R. Baker, Jr. directed directed a research stucy in which “BCTP,” presented experimental evidence that “BCTP” destroyed anthrax spores in a culture dish. Records further indicate “BCTP” was developed by someone at Novavax, Inc., Columbia, Maryland. In addition “The University of Michigan and Novavax had filed a patent application and there are plans to evaluated BCTP’s effectiveness against inhaled anthrax spores, as well as other bacteria and enveloped viruses.